A Dual-Plasmid CRISPR/Cas System for Mycotoxin Elimination in Polykaryotic Industrial Fungi.

Mycotoxin contamination causes disease and death in both humans and animals. Monascus Red, produced by Monascus purpureus, is used as a food colorant. However, its application is limited by contamination of the nephrotoxin citrinin, which is also produced by the fungus. Suppressing citrinin production by genetic engineering is difficult in a polykaryotic fungus such as M. purpureus. Hence, we developed a CRISPR/Cas system to delete large genomic fragments in polykaryotic fungi. Protoplast preparation and regeneration were optimized, and a dual-plasmid CRISPR/Cas system was designed to enable the deletion of the 15-kb citrinin biosynthetic gene cluster in M. purpureus industrial strain KL-001. The obtained homokaryotic mutants were stable, and citrinin was unambiguously eliminated. Moreover, the Monascus Red pigment production was increased by 2-5%. Our approach provides a powerful solution to solve this long-standing problem in the food industry and enables engineering of polykaryotic fungi for mycotoxin eliminations.